Thiolated nanocarriers for oral delivery of hydrophilic macromolecular drugs.

It was the aim of this study to investigate the effect of unmodified as well as thiolated anionic poly(acrylic acid) (PAA) and cationic chitosan (CS) utilized in free-soluble form and as nanoparticulate system on the absorption of the hydrophilic compound FD4 across intestinal epithelial cell layer with and without a mucus layer. Modifications of these polymers were achieved by conjugation with cysteine to PAA (PAA-Cys) and thioglycolic acid to CS (CS-TGA). Particles were prepared via ionic gelation and characterized based on their amount of thiol groups, particle size and zeta potential. Effects on the cell layer concerning absorption enhancement, transepithelial electrical resistance (TEER) and cytotoxicity were investigated. Permeation enhancement was evaluated with respect to in vitro transport of FD4 across Caco-2 cells, while mucoadhesion was indirectly examined in terms of adsorption behaviour when cells were covered with a mucus layer. Lyophilized particles displayed around 1000 µmol/g of free thiol groups, particle sizes of less than 300 nm and a zeta potential of 18 mV (CS-TGA) and -14 mV (PAA-Cys). Cytotoxicity studies confirmed that all polymer samples were used at nontoxic concentrations (0.5% m/v). Permeation studies revealed that all thiolated formulations had pronounced effects on the paracellular permeability of mucus-free Caco-2 layers and enhanced the permeation of FD4 3.0- to 5.3-fold. Moreover, polymers administered as particles showed a higher permeation enhancement than their corresponding solutions. However, the absorption-enhancing effect of each thiolated formulation was significantly (p<0.05) reduced when cells were covered with mucus layer. In addition, all formulations were able to decrease the TEER of the cell layer significantly (p<0.05). Therefore, both thiolated polymers as nanoparticulate delivery systems represent a promising tool for the oral administration of hydrophilic macromolecules.

Liposomes coated with thiolated chitosan enhance oral peptide delivery to rats.

The aim of the present study was the in vivo evaluation of thiomer-coated liposomes for an oral application of peptides. For this purpose, salmon calcitonin was chosen as a model drug and encapsulated within liposomes. Subsequently, the drug loaded liposomes were coated with either chitosan-thioglycolic acid (CS-TGA) or an S-protected version of the same polymer (CS-TGA-MNA), leading to an increase in the particle size of about 500 nm and an increase in the zeta potential from approximately -40 mV to a maximum value of about +44 mV, depending on the polymer. Coated liposomes were demonstrated to effectively penetrate the intestinal mucus layer where they came in close contact with the underlying epithelium. To investigate the permeation enhancing properties of the coated liposomes ex vivo, we monitored the transport of fluoresceinisothiocyanate-labeled salmon calcitonin (FITC-sCT) through rat small intestine. Liposomes coated with CS-TGA-MNA showed the highest effect, leading to a 3.8-fold increase in the uptake of FITC-sCT versus the buffer control. In vivo evaluation of the different formulations was carried out by the oral application of 40 µg of sCT per rat, either encapsulated within uncoated liposomes, CS-TGA-coated liposomes or CS-TGA-MNA-coated liposomes, or given as a solution serving as negative control. The blood calcium level was monitored over a time period of 24h. The highest reduction in the blood calcium level, to a minimum of 65% of the initial value after 6h, was achieved for CS-TGA-MNA-coated liposomes. Comparing the areas above curves (AAC) of the blood calcium levels, CS-TGA-MNA-coated liposomes led to an 8.2-fold increase compared to the free sCT solution if applied orally in the same concentration. According to these results, liposomes coated with S-protected thiomers have demonstrated to be highly valuable carriers for enhancing the oral bioavailability of salmon calcitonin.

Sexually dimorphic effects of GHRH on sleep-endocrine activity in patients with depression and normal controls - part I: the sleep eeg.

In patients with depression, enhanced secretion of ACTH and cortisol, a reduction in slow wave sleep (SWS) and a blunted nocturnal growth hormone (GH) surge have been described and attributed, at least partly, to an elevation of corticotropin-releasing hormone (CRH), hence a shift in the ratio between growth hormone-releasing hormone (GHRH) and CRH. We investigated the effects of pulsatile administration of GHRH (4x50 microgram, at hourly intervals between 2200 and 0100 h) on the sleep EEG (2300-0700 h) in patients with depression (16 females, 19 males, age range 19-76 years) and matched controls (20 females, 20 males). In patients compared with controls, NREM sleep and in particular stage 2 sleep was greatly decreased at baseline. GHRH treatment enhanced NREM sleep, and in particular stage 2 sleep in men, regardless of diagnosis, while decreasing it in women (F=6.0 and 7.1, p<0.05). In controls, aging was associated with a decrease in NREM sleep, including both SWS and stage 2 sleep (r= -0.45 r= -0.39, p<0.05), while in patients only SWS declined with age (r= -0.49, p<0.05). The significant decrease in NREM sleep including stage 2 sleep in patients with depression and elderly control subjects is compatible with the suggested role of sleep continuity and stage 2 sleep in cognitive functioning. GHRH promoted NREM sleep, stage 2 sleep and sleep continuity and might prove beneficial for improvement of cognitive function, at least in men. These data support the hypothesis that female gender, aging and depression are associated with a shift in the GHRH/CRH ratio towards CRH.

Aging does not affect the sleep endocrine response to total sleep deprivation in humans.

Aging is associated with decreased sleep continuity, slow wave sleep (SWS), growth hormone (GH) release and an increased hypothalamo-pituitary-adrenocortical (HPA) system activity. Total sleep deprivation (TSD) is a strong stimulus for sleep. To determine if aging affects the response to TSD, for the first time the combined effects of TSD on conventional and spectral sleep electroencephalographic (EEG) parameters and GH, cortisol and prolactin secretion were compared in elderly (60-80 years; n = 7) vs. younger subjects (20-30 years; n = 7). MANOVA revealed a reduction of SWS in the elderly. TSD led to an increase in SWS, a decrease in sleep onset latency, rapid eye movement (REM) density and by trend REM-latency without a global group difference. GH was reduced, whereas prolactin was enhanced in the elderly. After TSD GH was unchanged and prolactin secretion was enhanced without group difference. Thus, the plasticity of the sleep-endocrine system in response to TSD is sustained during aging. The possible involvement of the GABAergic system, that seems not to be severely impaired with age, is proposed.